Summary of work: Nuclear hormone receptors are ligand-activated factors modulating the expression of target genes. How these specific regulators interact with general transcription factors is unclear. It is believed that intermediary proteins-coactivators/corepressors could promote the interaction of these receptors with the transcriptional machinery.A number of non-histone chromosomal- or nuclear matrix proteins that had been originally described as structural proteins are reported to have transcription activities. Serendipitously, we have found that one of such proteins, hnRNP U (heterogeneous nuclear ribonucleoprotein U), exhibits a novel function as a Pol II elongation inhibitor. HnRNP U is known as a RNA- and the A/T-rich matrix attachment region DNA-binding protein. More than 70% of total hnRNP U is associated with nuclear matrix or chromatin, and half of the remaining 30% soluble fraction is present in the hnRNP particles. Accordingly, this protein has been proposed to participate in pre-mRNA processing and/or to play a role in the higher-order organization of chromatin. We have found that hnRNP U contains a region similar to the HIV-1 encoded Nef that is known to bind and inhibit various kinases and, through this Nef-like domain, HnRNP U specifically inhibits the kinase activities of TFIIH in the Pol II holoenzyme and thereby represses Pol II elongation. Transcription of a variety of cellular and viral genes including HIV-1 is regulated, at least in part, at the level of elongation. This is achieved by stimulating phosphorylation of the carboxyl terminal domain (CTD) of Pol II that consists of multiple repeats. One of the kinases that are thought to regulate CTD phosphorylation in vivo is TFIIH, a complex of nine subunit. Because the CTD in Pol II holoenzyme remains unphosphorylated despite the presence of TFIIH or other CTD kinase activities in the Pol II complex, it has been speculated that Pol II holoenzyme may be associated with an unknown inhibitor that negatively regulates CTD kinases. We have found that the middle domain of hnRNP U, which is dispensable for its interactions with nuclear matrix or hnRNP particles, targets hnRNP U to the Pol II holoenzyme complex where it inhibits Pol II elongation. Interestingly, the middle domain of hnRNP U contains a region similar to HIV-1 Nef which binds to and deregulates various kinases in the signal transduction pathways including serine/threonine kinases. Indeed, through the Nef-like domain, hnRNP U directly interacts with the catalytic subunit of TFIIH, Cdk7, and inhibits the kinase activity of TFIIH. Thus, we suggest that hnRNP U functions to downregulate CTD phosphorylation prior to productive elongation and with such function, becomes the first characterized elongation inhibitor in the Pol II holoenzyme. Clone 6. Initially, we obtained about 650 bp of this novel cDNA. This sequence was used to probe multiple tissues blot. Northern analysis revealed that this message is exclusively expressed at a high level in the rat testis and embryonic brain. Alternative splicing at the 5' end generated two mRNA forms of 2.3 and 1.5 kb. Full length cDNA clones were obtained by screening a rat testis cDNA library. DNA sequence analysis showed that this novel protein contains several repeats of novel leucine-rich motif (LXXLL), a signature of known transcription co-activators. It is likely that this protein might be a testis specific co-activator/co-repressor of estrogen receptor. Recently, we provided further evidence of Clone 6 and estrogen receptor interaction using a glutathione-S-transferase assay, which demonstrated that a fusion protein (clone 6) associated with estrogen receptor in an estrogen dependent fashion. Presently, interaction of other nuclear hormone receptors with the clone 6 are being investigated.